PROJECT SUMMARY Disorders of transepithelial ion transport underlie clinical disorders of extracellular volume, blood pressure, and electrolytes, but molecular mechanisms of transepithelial ion transport are difficult to directly examine in the mammalian nephron. The applicants' long-term goal is to better understand epithelial ion transport mechanisms relevant to human kidney function, in sufficient molecular detail to define new therapeutic strategies. The overall objective of this application is to identify regulators of a kinase cascade, consisting of WNK (With No Lysine) and SPAK/OSR1 (Ste20-related proline alanine rich kinase/oxidative stress response) kinases, that plays an essential role in sodium and potassium homeostasis through the regulation of renal transepithelial ion transport. The application builds on three recent findings: Cl- directly binds to the WNK kinase domain to inhibit autophosphorylation and activation; the scaffold protein Mo25 (Mouse protein 25/Cab39) enhances the activity of SPAK/OSR1; and low potassium diet activates WNK-SPAK/OSR1 signaling. The central hypothesis is that transepithelial ion flux is directly regulated by transported ions (Cl- and K+) through modulation of WNK-SPAK/OSR1 signaling, while Mo25 provides additional regulatory control. The rationale is that better understanding of these molecular mechanisms will allow the design of novel therapeutics with fewer off-target effects. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the roles of Cl- and K+ in the regulation of WNK isoforms in transepithelial ion transport; 2) Determine the role of Mo25 in WNK signaling in a transporting epithelium; and 3) Probe tubule physiology using newly developed chemical WNK inhibitors. The approach is innovative by bridging fundamental molecular insights gained from biophysical studies, with the functional physiological roles of those molecular mechanisms, using newly developed platforms and tools to probe questions of transporting epithelium biology. Assays have been established, and demonstrated feasible in the investigators' hands, to examine regulation of Drosophila and mammalian WNKs by Cl- and K+ in vitro and in the fly renal tubule, and to measure intracellular Cl- in live tubules, with temporal resolution; and to measure transepithelial ion flux in genetically modified, or pharmacologically treated, tubules. The proposed research is significant, because it is expected to advance understanding of molecular mechanisms of WNK-SPAK/OSR1 regulation in a transporting renal epithelium. The studies will determine: 1) how quickly changes in intracellular Cl- change WNK activity; 2) whether WNKs act as K+ sensors; and 3) the role of Mo25 in transepithelial ion transport. In addition, these studies will further develop recently identified pharmacological WNK inhibitors, which will be a useful tool for further probing the biology of WNK-SPAK/OSR1 signaling in Drosophila and mammalian systems, and potentially serve as the basis for future development of therapeutic compounds for the treatment of volume overload, hypertension and hyperkalemia.